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PubMed1992

Role of estrogen receptor variants in the development of hormone resistance in breast cancer.

Recent evidence suggests that the progression to hormone resistance in some breast tumors is due to mutations in the estrogen receptor (ER). Various types of ER variants have been found in breast cancer biopsies and breast cancer cell lines. The ER variants include dominant-positive receptors that are transcriptionally active in the absence of estrogen, and dominant-negative receptors that are themselves transcriptionally inactive but prevent the action of the normal receptor. The mechanisms by which these variants cause loss of hormonal control is becoming clear. ER variants may be prognostic factors for breast cancer. By modifying the action of ER variants, it should be possible to develop new strategies for treatment of malignant breast disease.

Animals↗

PubMed1994

Rapid protection against human immunodeficiency virus type 1 (HIV-1) replication mediated by high efficiency non-retroviral delivery of genes interfering with HIV-1 tat and gag.

Efficient transduction of inhibitory genes is a critical requirement in the development of a gene therapy strategy against human immunodeficiency virus type 1 (HIV-1). Commonly used systems based on retrovirus-mediated gene delivery are characterized by low efficiency gene transfer into the target cell. Genes were transduced in the absence of cell selection into 60-90% of human CD4+ cells by using a novel technique that allows high efficiency gene transfer mediated by adenoviruses coupled with DNA-polylysine complexes. Protection of these cells against HIV-1 acute infection was evaluated by transducing them with three different inhibitory genes which interfere with HIV-1 replication at separate levels (polymeric Tat activation response element [TAR] decoy, dominant-negative mutant of the gag gene and antisense sequences of the gag gene) and subsequent challenging with HIV-1. The polymeric TAR decoy inhibited HIV-1 replication over 95%. Both the dominant-negative mutant and the antisense sequence of the gag gene were less potent inhibitors than the polymeric-TAR decoy. Combinations of either polymeric-TAR with dominant-negative mutant or antisense of the gag gene synergistically enhanced the inhibitory effects of the single genes. These data suggest that the combination of a highly efficient transduction technique with effective HIV-1 inhibitory genes confers rapid protection against HIV-1 acute infection in vitro.

Adenoviridae↗

PubMed1991

Characterization of bovine papillomavirus E1 region deletion mutants associated with neoplastic transformation in a murine cell line.

Spontaneous focus formation in the contact-inhibited C127 cell line, cl.2, harbouring multiple copies of a bovine papillomavirus type 1 deletion mutant, was associated with the evolution of further viral genomic deletions in addition to an amplification of the viral genome copy number. Three simple frameshift deletions of 308, 605 and 1291 bp, associated with separate transformation events, were mapped within the E1 open reading frame, implying a common mechanism of spontaneous transformation in this cell line. Furthermore, each transformed cell line also retained multiple copies of the intact E1 gene, suggesting that these novel deletion mutants might function by a dominant-negative mechanism to disrupt the normal control of viral DNA replication or viral transcription. These mutants had the potential to encode truncated E1 polypeptides with a common N-terminal region encoded by the 5' end of E1, i.e. overlapping the previously described E1 modulator gene. A possible role for these mutants in diverting a lethal type of virus-cell interaction is discussed.

Animals↗

PubMed1991

The type I human T-cell leukemia virus (HTLV-I) Rex trans-activator binds directly to the HTLV-I Rex and the type 1 human immunodeficiency virus Rev RNA response elements.

The Rex protein of the type I human T-cell leukemia virus (HTLV-I) is essential for the replication of this pathogenic retrovirus and, surprisingly, can also replace the function of the structurally distinct Rev protein of the type 1 human immunodeficiency virus (HIV-1). Rex action requires a 255-nucleotide viral RNA stem-loop structure termed the Rex RNA response element (RexRE) located in the 3' retroviral long terminal repeat. Rex function leads to the induced cytoplasmic expression of the incompletely spliced family of viral mRNAs that uniquely encode the HTLV-I structural and enzymatic proteins (Gag, Pol, and Env). Our studies now demonstrate that Rex acts by binding directly to the RexRE in a sequence-specific manner. These effects of Rex require the presence of a 10-nucleotide subregion of the RexRE that is essential for Rex function in vivo. Dominant-negative mutants of Rex also bind to the RexRE with high affinity, while a recessive-negative Rex mutant altered within its arginine-rich, positively charged domain fails to engage the RexRE. Analogously, both the wild-type and dominant-negative Rex proteins specifically bind to the structurally distinct HIV-1 Rev response element, a finding that likely underlies the respective stimulatory and inhibitory effects of these HTLV-I proteins in the heterologous HIV-1 system. However, consistent with their lack of amino acid homology, the binding sites for Rex and Rev within the HIV-1 Rev response element are distinct.

Binding Sites↗

PubMed1995

CDC42 and Rac1 control different actin-dependent processes in the Drosophila wing disc epithelium.

Cdc42 and Rac1 are members of the rho family of small guanosinetriphosphatases and are required for a diverse set of cytoskeleton-membrane interactions in different cell types. Here we show that these two proteins contribute differently to the organization of epithelial cells in the Drosophila wing imaginal disc. Drac1 is required to assemble actin at adherens junctions. Failure of adherens junction actin assembly in Drac1 dominant-negative mutants is associated with increased cell death. Dcdc42, on the other hand, is required for processes that involve polarized cell shape changes during both pupal and larval development. In the third larval instar, Dcdc42 is required for apico-basal epithelial elongation. Whereas normal wing disc epithelial cells increase in height more than twofold during the third instar, cells that express a dominant-negative version of Dcdc42 remain short and are abnormally shaped. Dcdc42 localizes to both apical and basal regions of the cell during these events, and mediates elongation, at least in part, by effecting a reorganization of the basal actin cytoskeleton. These observations suggest that a common cdc42-based mechanism may govern polarized cell shape changes in a wide variety of cell types.

Actins↗

PubMed1992

Serum-, TPA-, and Ras-induced expression from Ap-1/Ets-driven promoters requires Raf-1 kinase.

Raf-1 serine-threonine protein kinase has the hallmarks of a critical switch that connects growth factor receptor activation at the cell membrane with transcriptional events in the nucleus. We show by use of Raf-1 dominant-negative mutants that Raf-1 is required for serum-, TPA-, and Ras-induced expression from the oncogene-responsive element in the polyomavirus enhancer. The minimal region of Raf-1 that displays this dominant-negative phenotype (Raf-C4) contains a cysteine finger motif. Raf-C4 appears to function by titrating out a Raf-1-activating factor that is induced by Ras following serum or TPA treatment of NIH-3T3 cells. In addition, we show that Raf-1 and Ras cooperate in trans-activation through the oncogene-responsive element and that the cysteine-rich region is necessary for this effect.

3T3 Cells↗

PubMed1995

Basal expression of the cystic fibrosis transmembrane conductance regulator gene is dependent on protein kinase A activity.

The cystic fibrosis transmembrane conductance regulator (CFTR) functions as a Cl- channel that becomes activated after phosphorylation by cAMP-dependent protein kinase (PKA). We demonstrate that PKA also plays a crucial role in maintaining basal expression of the CFTR gene in the human colon carcinoma cell line T84. Inhibition of PKA activity by expression of a dominant-negative regulatory subunit or treatment with the PKA-selective inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89) caused a complete suppression of CFTR gene expression without affecting other constitutively active genes. Basal expression of a 2.2-kb region of the CFTR promoter linked to a luciferase reporter gene (CFTR-luc) exhibited the same dependence on PKA. The ability of cAMP to induce CFTR over basal levels is cell-type specific. In T84 cells, both the endogenous CFTR gene and CFTR-luc exhibited only a modest inducibility (approximately 2-fold), whereas in the human choriocarcinoma cell line JEG-3, CFTR-luc could be induced at least 4-fold. A variant cAMP-response element is present at position -48 to -41 in the CFTR promoter, and mutation of this sequence blocks basal expression. We conclude that cAMP, acting through PKA, is an essential regulator of basal CFTR gene expression and may mediate an induction of CFTR in responsive cell types.

Base Sequence↗

PubMed1995

Regulation of cell signaling by the cytoplasmic domains of the heat-stable enterotoxin receptor: identification of autoinhibitory and activating motifs.

Infection with enterotoxigenic Escherichia coli is a leading cause of traveler's diarrhea. Many enterotoxigenic E. coli strains produce heat-stable enterotoxin (ST), a peptide that binds to the intestinal receptor guanylyl cyclase C known as STaR. The toxin-receptor interaction elevates intracellular cGMP, which then activates apical chloride secretion, resulting in secretory diarrhea. In this report, we examine how the intracellular domains of STaR participate in the propagation and regulation of signaling. We show that STaR exists as an oligomer in both the presence and the absence of toxin. We also demonstrate that deletion of the intracellular kinase-homology domain produces a constitutively active mutant, suggesting that this domain subserves an autoinhibitory function. Finally, we constructed a point mutant within a highly conserved region of the cyclase domain that completely inactivates the catalytic activity of guanylyl cyclase. Cotransfection of this point mutant with wild-type receptor causes a dominant-negative effect on receptor activation. This suggests that interaction of receptor subunits is required for toxin-induced activation and that the cyclase domain is involved in this essential interaction. We propose that the binding of ST to STaR promotes a conformational change across the cell membrane. This removes the inhibitory effects of the kinase-homology domain and promotes an interaction between cyclase domains that leads to receptor activation. The data suggest a paradigm of signal transduction that may also be relevant to other members of the guanylyl cyclase receptor family.

Amino Acid Sequence↗

PubMed1995

Disruption of gastrulation movements in Xenopus by a dominant-negative mutant for C-cadherin.

Gastrulation in Xenopus laevis transforms a ball of undifferentiated cells into a well-organized elongated embryo with a distinct body axis, as a result of extensive cell rearrangements and movements. Since cell adhesion is thought to play an important role during tissue morphogenesis, we investigated the potential role for C-cadherin, a primary cell-cell adhesion molecule in Xenopus, in gastrulation movements. A deletion mutant consisting of the extracellular and transmembrane domains but lacking the cytoplasmic tail (Ctrunc) was constructed to act as a dominant-negative inhibitor of the endogenous protein. Injection of in vitro transcribed Ctrunc mRNA into the prospective dorsal involuting marginal zone, the region that undergoes the most extensive movements, caused a reproducible defect in gastrulation. The defect, an inability to complete involution and close the blastopore, was cadherin-specific, because it could be rescued by expressing full length C-cadherin. Ctrune also seems to inhibit morphogenetic movements of animal cap explants isolated from injected embryos. Expression of Ctrunc did not appear to interfere with inductive processes in the embryos that developed gastrulation defects, because they formed complete axial structures, including a complete head and notochord. These results demonstrate that C-cadherin plays a critical role in Xenopus laevis gastrulation movements, and they support the hypothesis that cadherins can mediate cellular rearrangements during tissue morphogenesis.

Animals↗

PubMed1991

Requirement for acidic amino acid residues immediately N-terminal to the conserved domain of Saccharomyces cerevisiae TFIID.

TFIID binds to TATA boxes and initiates the assembly of general transcription factors and pol II on promoters. TFIID proteins from various species consist of a highly conserved carboxy terminal domain and very divergent amino terminal domains. We investigated the function of the non-conserved amino terminal domain (residues 1-60) of Saccharomyces cerevisiae TFIID (YIID, 240 residues) by testing the ability of a series of YIID amino terminal deletion mutants to complement a YIID deficient yeast strain. Mutants with deletions up to amino acid 48 restored the YIID deficient yeast strain to an apparently wild type phenotype. However, deletion up to position 57 or 60 produced yeast strains which formed extremely small colonies. Moreover, overexpression of YIID delta 2-57 or YIID delta 3-60 protein in the presence of wild type YIID resulted in a dominant-negative inhibition of growth. No difference between the basal transcriptional activity of wild type YIID and these amino terminal deletion mutants was observed in vitro. However, transcriptional activation in vivo of promoter-lacZ fusions showed that the YIID delta 2-57 deletion affects the ability of certain promoters (CUP1 and an HSP UAS-CYC1 promoter hybrid promoter) to respond to upstream factor stimulation. At least one inducible promoter, PHO5, was not affected by this deletion. The defect produced by YIID delta 2-57 was due to the deletion of several acidic residues present between residues 48 and 57. The results show that the conserved carboxy terminal domain of YIID is sufficient for cell viability. However, an acidic region just amino terminal to the conserved domain is required for normal growth and transcription control in most yeast strains.

Amino Acid Sequence↗

PubMed1995

Delayed assembly of desmosomes in keratinocytes with disrupted classic-cadherin-mediated cell adhesion by a dominant negative mutant.

We examined whether classic cadherins play a role in the formation of desmosomes using a mouse keratinocyte, PAMcN390 delta cell, which shows disrupted classic-cadherin-mediated cell adhesion by introduction of a dominant-negative mutant of N-cadherin. The expression of the mutant did not alter that of endogenous E-cadherin or desmoplakin. In control cells with functional classic cadherins, we observed redistribution of desmoplakin to cell-cell borders with insertions of keratin filaments at the contact sites as soon as 2 h after calcium elevation, after an earlier event of E-cadherin translocation to the cell-cell contact sites. In contrast, in the PAMcN390 delta cells, which showed retarded translocation of E-cadherin, the redistribution of desmoplakin and the rearrangement of keratin filaments were delayed as late as 24 h after the calcium elevation. The acquisition of Nonidet P-40 insolubility of desmoplakins also was found to be delayed in the PAMcN390 delta cells. These findings indicate that the disruption of classic cadherin affected the organization of desmosomes upon calcium elevation and suggest that the proper function of classic cadherins is a prerequisite for desmosome assembly in keratinocytes.

Animals↗

PubMed1992

Insulin/IGF-1 hybrid receptors: implications for the dominant-negative phenotype in syndromes of insulin resistance.

Classical insulin and IGF-1 receptors are alpha 2 beta 2 heterotetrameric complexes synthesized from two identical alpha beta half-receptor precursors. Recent data strongly suggests, however, that nonidentical alpha beta half-receptor precursors can assemble to generate hybrid holoreceptor species both in vivo and in vitro. This review focuses primarily on two types of hybrid receptors. The first type is an insulin/IGF-1 hybrid receptor generated by the association of an alpha beta insulin half-receptor with an alpha beta IGF-1 half-receptor. The second type is one formed from a wildtype (kinase-active) insulin or IGF-1 alpha beta half-receptor and a mutant (kinase-inactive) insulin alpha beta half-receptor. Although the functional properties of insulin/IGF-1 hybrid receptors have not yet been completely defined, wildtype/mutant hybrid receptors are essentially substrate kinase inactive. These data indicate that the mutant alpha beta half-receptor exerts a transdominant inhibition upon the wildtype alpha beta half-receptor within the alpha 2 beta 2 holoreceptor complex. This defect in substrate kinase activity may contribute to the molecular defect underlying some syndromes of severe insulin resistance and diabetes. Heterozygous individuals expressing both wildtype and mutant tyrosine kinase-defective insulin receptor precursors demonstrate varying degrees of insulin resistance and diabetes. In addition, cell lines which express both endogenous wildtype and transfected kinase-defective insulin receptors display markedly decreased insulin and IGF-1 sensitivity and responsiveness. Formation of hybrid receptors which results in premature termination of insulin signal transduction may be one mechanism underlying the observation that kinase-inactive receptors inhibit the function of native receptors.

Animals↗

PubMed2025

Diagnostic Implications and Correlates of Plasma Adenosine Deaminase 2 Activity and ADA2 Variants.

OBJECTIVE: Deficiency of adenosine deaminase 2 (DADA2) is a monogenic autoinflammatory disease manifested as polyarteritis nodosa, stroke, and bone marrow failure. Leveraging an international cohort of 200 DADA2 cases, we aimed to characterize the diagnostic utility of a plasma ADA2 enzyme activity assay and understand the implications of residual ADA2 activity. METHODS: Data were collected from individuals who underwent ADA2 testing from 2018 to 2025. Plasma ADA2 activity was determined using an established spectrophotometric assay. ADA2 variants were analyzed in transfected cells by enzyme assay and western blotting. RESULTS: We determined that plasma ADA2 activity is 99.0% and 96.0% sensitive and 99.7% and 98.8% specific in distinguishing genetically confirmed DADA2 cases from controls and carriers, respectively. Eighteen individuals with DADA2 (9%) possessed detectable ADA2 activity, including several cases with levels seen in carriers. Residual ADA2 activity was associated with the vasculitis/inflammatory phenotype but not with disease severity. Genotype analysis revealed that 14 of 18 cases with residual plasma activity possessed at least one hypomorphic missense variant with greater than 20% residual ADA2 function when overexpressed in 293T cells, often occurring in trans with a more deleterious variant. In vitro analysis revealed that missense ADA2 variants exert variable dominant-negative effects by forming large intracellular protein aggregates via disulfide bond formation at a cysteine residue (Cys408). CONCLUSION: We confirmed the utility of plasma ADA2 activity as a diagnostic assay and showed that the inflammatory phenotype of DADA2 occurred in cases with residual activity. In vitro findings illustrate potential interactions of ADA2 variants to synergistically disrupt protein function.

Humans↗

PubMed1995

Dependence of globin gene expression in mouse erythroleukemia cells on the NF-E2 heterodimer.

High-level, tissue-specific expression of the beta-globin genes requires the presence of an upstream locus control region (LCR). The overall enhancer activity of the beta-globin complex LCR (beta-LCR) is dependent on the integrity of the tandem NF-E2 sites of HS-2. The NF-E2 protein which binds these sites is a heterodimeric basic leucine zipper protein composed of a tissue-specific subunit, p45 NF-E2, and a smaller subunit, p18 NF-E2, that is widely expressed. In these studies, we sought to investigate the role of NF-E2 in globin expression. We show that expression of a dominant-negative mutant p18 greatly reduces the amount of functional NF-E2 complex in the cell. Reduced levels of both alpha- and beta-globin were associated with the lower levels of NF-E2 activity in this cell line. Globin expression was fully restored upon the introduction of a tethered p45-p18 heterodimer. We also examined CB3 cells, a mouse erythroleukemia (MEL) cell line that does not express endogenous p45 NF-E2, and demonstrated that the restoration of globin gene expression was dependent upon the levels of expressed tethered NF-E2 heterodimer. Results of DNase I hypersensitivity mapping and in vivo footprinting assays showed no detectable chromatin alterations in beta-LCR HS-2 due to loss of NF-E2. Finally, we examined the specificity of NF-E2 for globin gene expression in MEL cells. These experiments indicate a critical role for the amino-terminal domain of p45 NF-E2 and show that a related protein, LCRF1, is unable to restore globin gene expression in p45 NF-E2-deficient cells. From these results, we conclude that NF-E2 is specifically required for high level goblin gene expression in MEL cells.

Animals↗

PubMed2025

DNA-binding affinity and specificity determine the phenotypic diversity in BCL11B-related disorders.

BCL11B is a Cys2-His2 zinc-finger (C2H2-ZnF) domain-containing, DNA-binding, transcription factor with established roles in the development of various organs and tissues, primarily the immune and nervous systems. BCL11B germline variants have been associated with a variety of developmental syndromes. However, genotype-phenotype correlations along with pathophysiologic mechanisms of selected variants mostly remain elusive. To dissect these, we performed genotype-phenotype correlations of 92 affected individuals harboring a pathogenic or likely pathogenic BCL11B variant, followed by immune phenotyping, analysis of chromatin immunoprecipitation DNA-sequencing data, dual-luciferase reporter assays, and molecular modeling. These integrative analyses enabled us to define three clinical subtypes of BCL11B-related disorders. It is likely that gene-disruptive BCL11B variants and missense variants affecting zinc-binding cysteine and histidine residues cause mild to moderate neurodevelopmental delay with increased propensity for behavioral and dental anomalies, allergies and asthma, and reduced type 2 innate lymphoid cells. Missense variants within C2H2-ZnF DNA-contacting α helices cause highly variable clinical presentations ranging from multisystem anomalies with demise in the first years of life to late-onset, hyperkinetic movement disorder with poor fine motor skills. Those not in direct DNA contact cause a milder phenotype through reduced, target-specific transcriptional activity. However, missense variants affecting C2H2-ZnFs, DNA binding, and "specificity residues" impair BCL11B transcriptional activity in a target-specific, dominant-negative manner along with aberrant regulation of alternative DNA targets, resulting in more severe and unpredictable clinical outcomes. Taken together, we suggest that the phenotypic severity and variability is largely dependent on the DNA-binding affinity and specificity of altered BCL11B proteins.

Adolescent↗

PubMed1990

fos-jun conspiracy: implications for the cell.

Two nuclear oncoproteins, fos and jun (AP-1), cooperate in forming a very stable heterodimeric complex that binds to the AP-1 site on DNA with high affinity. The 'leucine zipper' domain of both fos and jun is necessary for the formation of this heterodimer. Mutations of single residues within the leucine zipper domain have no effect on protein complex formation. However, results from mutagenesis of the first leucine of the heptad repeat in either fos or jun basic regions and alteration of the spacing between the basic and leucine zipper domains indicate that the basic region of fos plays a crucial role in determining the DNA binding affinity of the transcriptional complex. Mutations of the basic amino acids in fos protein prevent binding to the tumour promoter response element (TRE) in the presence of wild-type jun protein. Thus fos protein appears to be dominant in jun-fos binding to DNA, even though fos alone cannot bind to TRE. Mutants in the basic region of fos and jun can be exploited as dominant-negative mutants to ablate the normal fos cellular function.

Amino Acid Sequence↗

PubMed1992

Dominant-negative mutants of a yeast G-protein beta subunit identify two functional regions involved in pheromone signalling.

The STE4 gene, which encodes the beta subunit of the mating response G-protein in the yeast Saccharomyces cerevisiae, was subjected to a saturation mutagenesis using 'doped' oligodeoxynucleotides. We employed a genetic screen to select dominant-negative STE4 mutants, which when overexpressed from the GAL1 promoter, interfered with the signalling function of the wild type protein. The identified inhibitory amino acid alterations define two small regions that are crucially involved in transmitting the mating signal from G beta to downstream components of the signalling pathway. These results underline the positive signalling role of yeast G beta and assign for the first time the positive signalling function of a G-protein beta subunit to specific structural features.

Amino Acid Sequence↗

PubMed1993

Targeted expression of a dominant-negative FGF receptor mutant in the epidermis of transgenic mice reveals a role of FGF in keratinocyte organization and differentiation.

In this study we used a dominant-negative FGF receptor mutant to block FGF function in a specific tissue of transgenic mice. The mutant receptor, which is known to block signal transduction in cells when co-expressed with wild-type receptors, was targeted to suprabasal keratinocytes using a keratin 10 promoter. The transgene was expressed specifically in the skin and highest expression levels were found in the tail. Expression of the mutant receptor disrupted the organization of epidermal keratinocytes, induced epidermal hyperthickening and resulted in an aberrant expression of keratin 6. This suggests that FGF is essential for the morphogenesis of suprabasal keratinocytes and for the establishment of the normal program of keratinocyte differentiation. Our study demonstrates that dominant-negative growth factor receptors can be used to block selectively the action of a growth factor in specific tissues of transgenic mice.

Animals↗